Colloidal gold chromatography kit for nucleic acid detection of novel coronaviruses (2019-nCoV) and application thereof

Abstract

The invention discloses a colloidal gold chromatography kit for detecting novel coronaviruses (2019-nCoV) and an application thereof. According to the kit, a collected sample is cracked by a cell lysis solution to release pathogen nucleic acid, and then under the action of reverse transcriptase and T7RNA polymerase, amplification of pathogen nucleic acid fragments is realized through reverse transcription and transcription processes. An amplified RNA product is recognized and captured by a specific probe in a detection solution to form an RNA amplification product-specific probe-gold probe compound, and the compound is fixed on an NC membrane through lateral flow chromatography to form a visible strip, so that the detection of the pathogen nucleic acid is realized. The kit has no RNA extraction process, does not need a special instrument, has difficulty in pollution in actual detection based on RNA isothermal amplification, has the advantages of high sensitivity, high specificity and simplicity in operation, and makes wide application of novel coronavirus (2019-nCoV) nucleic acid detection possible.

Description

technical field

[0001] The invention relates to the technical field of biological detection, in particular to a colloidal gold chromatography kit for detection of novel coronavirus (2019-nCoV) and its application. Background technique

[0002] Coronaviruses are non-segmented, single-stranded positive-sense RNA viruses belonging to the order Nidovirales, Coronaviridae, and Orthocoronavirinae, which are divided into subfamilies according to serotype and genomic characteristics. α, β, γ, and δ genera. There are 6 coronaviruses known to infect humans, including 229E and NL63 of the genus α, OC43 and HKU1 of the genus β, Middle East respiratory syndrome-associated coronavirus (MERSr-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV). CoV). The novel coronavirus 2019-nCoV is a novel coronavirus belonging to the genus Beta. At present, there is relatively little knowledge of its physical and chemical properties, and most of the knowledge of related pro...

Examples

Embodiment 1

[0098] [Example 1] Preparation of nucleic acid detection test strips

[0099] The main raw materials required for the preparation of nucleic acid detection test strips: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC bottom plate, etc.

[0100] 1. Spray film:

[0101] Detection line E-T line: capable of capturing and binding the new coronavirus (2019-nCoV) E gene-specific probe CES sequence, (10μM), spray film volume: 2-3μL / cm;

[0102] Detection line ORF1ab-T line: capable of capturing and binding the CES sequence of the novel coronavirus (2019-nCoV) ORF1ab gene-specific probe (10μM), spray volume: 2-3μL / cm;

[0103] Detection line internal reference-T line: capable of capturing and binding internal reference specific probe CES sequence (10μM), spray film volume: 2~3μL / cm;

[0104] Quality control line (C line): able to capture and bind to the C-line chromogenic probe sequence (10μM), spray film volume: 2-3μL / cm;

[0105] After spraying the film, it...

Embodiment 2

[0108] [Example 2] Sensitivity Test

[0109] Select a known concentration of the pseudovirus containing the 2019-nCoV target gene, perform a 10-fold concentration gradient dilution, and repeat each gradient dilution three times separately, and use the lowest dilution concentration with a 100% positive detection rate as the estimated detection limit , after the estimated detection limit is determined, dilute the 2019-nCoV pseudovirus to near the estimated detection limit concentration, and use the kit for detection, each concentration is tested 20 times to further accurately determine the minimum detection limit concentration (the positive rate is selected at The dilution above 95% is used as the detection limit sensitivity of this kit).

[0110] Table 1. 2019-nCoV Pseudovirus Experimental Results - Determination of Estimated Detection Limits

[0111]

[0112] Table 2. Experimental results of 2019-nCoV pseudovirus - determination of detection limit

[0113]

[0114] ...

Embodiment 3

[0116] [Example 3] Specificity Verification

[0117] Other pathogens that are similar to the 2019 novel coronavirus species or cause similar symptoms (such as seasonal influenza A H1N1 virus, novel influenza A H1N1 (2009) virus, influenza A H3N2, H5N1, H7N9, influenza B Yamagata, influenza B Influenza type Victoria, RSV type A, RSV type B, parainfluenza type I, parainfluenza type II, parainfluenza type III, rhinovirus group A, B, C, adenovirus 1, 2, 3, 4, Types 5, 7, 55, enterovirus A, B, C, D, human metapneumovirus (human metapneumovirus), Epstein-Barr virus, measles virus, human cytomegalovirus, rotavirus, norovirus, Mumps virus, varicella-zoster virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella, Bordetella pertussis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, tuberculosis Mycobacterium, Aspergillus fumigatus, Candida albicans, Candida glabrata, Cryptococcus neoformans, coronavirus (HK...

technical field

[0001] The invention relates to the technical field of biological detection, in particular to a colloidal gold chromatography kit for detection of novel coronavirus (2019-nCoV) and its application. Background technique

[0002] Coronaviruses are non-segmented, single-stranded positive-sense RNA viruses belonging to the order Nidovirales, Coronaviridae, and Orthocoronavirinae, which are divided into subfamilies according to serotype and genomic characteristics. α, β, γ, and δ genera. There are 6 coronaviruses known to infect humans, including 229E and NL63 of the genus α, OC43 and HKU1 of the genus β, Middle East respiratory syndrome-associated coronavirus (MERSr-CoV) and severe acute respiratory syndrome-associated coronavirus (SARSr-CoV). CoV). The novel coronavirus 2019-nCoV is a novel coronavirus belonging to the genus Beta. At present, there is relatively little knowledge of its physical and chemical properties, and most of the knowledge of related pro...

Examples

Embodiment 1

[0098] [Example 1] Preparation of nucleic acid detection test strips

[0099] The main raw materials required for the preparation of nucleic acid detection test strips: nitrocellulose membrane (NC membrane), sample pad, absorbent paper, PVC bottom plate, etc.

[0100] 1. Spray film:

[0101] Detection line E-T line: capable of capturing and binding the new coronavirus (2019-nCoV) E gene-specific probe CES sequence, (10μM), spray film volume: 2-3μL / cm;

[0102] Detection line ORF1ab-T line: capable of capturing and binding the CES sequence of the novel coronavirus (2019-nCoV) ORF1ab gene-specific probe (10μM), spray volume: 2-3μL / cm;

[0103] Detection line internal reference-T line: capable of capturing and binding internal reference specific probe CES sequence (10μM), spray film volume: 2~3μL / cm;

[0104] Quality control line (C line): able to capture and bind to the C-line chromogenic probe sequence (10μM), spray film volume: 2-3μL / cm;

[0105] After spraying the film, it...

Embodiment 2

[0108] [Example 2] Sensitivity Test

[0109] Select a known concentration of the pseudovirus containing the 2019-nCoV target gene, perform a 10-fold concentration gradient dilution, and repeat each gradient dilution three times separately, and use the lowest dilution concentration with a 100% positive detection rate as the estimated detection limit , after the estimated detection limit is determined, dilute the 2019-nCoV pseudovirus to near the estimated detection limit concentration, and use the kit for detection, each concentration is tested 20 times to further accurately determine the minimum detection limit concentration (the positive rate is selected at The dilution above 95% is used as the detection limit sensitivity of this kit).

[0110] Table 1. 2019-nCoV Pseudovirus Experimental Results - Determination of Estimated Detection Limits

[0111]

[0112] Table 2. Experimental results of 2019-nCoV pseudovirus - determination of detection limit

[0113]

[0114] ...

Embodiment 3

[0116] [Example 3] Specificity Verification

[0117] Other pathogens that are similar to the 2019 novel coronavirus species or cause similar symptoms (such as seasonal influenza A H1N1 virus, novel influenza A H1N1 (2009) virus, influenza A H3N2, H5N1, H7N9, influenza B Yamagata, influenza B Influenza type Victoria, RSV type A, RSV type B, parainfluenza type I, parainfluenza type II, parainfluenza type III, rhinovirus group A, B, C, adenovirus 1, 2, 3, 4, Types 5, 7, 55, enterovirus A, B, C, D, human metapneumovirus (human metapneumovirus), Epstein-Barr virus, measles virus, human cytomegalovirus, rotavirus, norovirus, Mumps virus, varicella-zoster virus, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella, Bordetella pertussis, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Klebsiella pneumoniae, tuberculosis Mycobacterium, Aspergillus fumigatus, Candida albicans, Candida glabrata, Cryptococcus neoformans, coronavirus (HK...

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